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22 pages, 20414 KiB

Open AccessArticle

Two Distinct Modes of DNA Binding by an MCM Helicase Enable DNA Translocation

by Martin Meagher, Alexander Myasnikov and Eric J. Enemark

Int. J. Mol. Sci. 2022, 23(23), 14678; https://doi.org/10.3390/ijms232314678 - 24 Nov 2022

Cited by 1 | Viewed by 2335

Abstract

A six-subunit ATPase ring forms the central hub of the replication forks in all domains of life. This ring performs a helicase function to separate the two complementary DNA strands to be replicated and drives the replication machinery along the DNA. Disruption of [...] Read more.

A six-subunit ATPase ring forms the central hub of the replication forks in all domains of life. This ring performs a helicase function to separate the two complementary DNA strands to be replicated and drives the replication machinery along the DNA. Disruption of this helicase/ATPase ring is associated with genetic instability and diseases such as cancer. The helicase/ATPase rings of eukaryotes and archaea consist of six minichromosome maintenance (MCM) proteins. Prior structural studies have shown that MCM rings bind one encircled strand of DNA in a spiral staircase, suggesting that the ring pulls this strand of DNA through its central pore in a hand-over-hand mechanism where the subunit at the bottom of the staircase dissociates from DNA and re-binds DNA one step above the staircase. With high-resolution cryo-EM, we show that the MCM ring of the archaeal organism Saccharolobus solfataricus binds an encircled DNA strand in two different modes with different numbers of subunits engaged to DNA, illustrating a plausible mechanism for the alternating steps of DNA dissociation and re-association that occur during DNA translocation. Full article

(This article belongs to the Special Issue Mechanisms of DNA Replication Fork Progression, Stalling, and Rescue)

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15 pages, 4151 KiB

Open AccessArticle

DNA Polymerase-Parental DNA Interaction Is Essential for Helicase-Polymerase Coupling during Bacteriophage T7 DNA Replication

by Chen-Yu Lo and Yang Gao

Int. J. Mol. Sci. 2022, 23(3), 1342; https://doi.org/10.3390/ijms23031342 - 25 Jan 2022

Cited by 3 | Viewed by 4040

Abstract

DNA helicase and polymerase work cooperatively at the replication fork to perform leading-strand DNA synthesis. It was believed that the helicase migrates to the forefront of the replication fork where it unwinds the duplex to provide templates for DNA polymerases. However, the molecular [...] Read more.

DNA helicase and polymerase work cooperatively at the replication fork to perform leading-strand DNA synthesis. It was believed that the helicase migrates to the forefront of the replication fork where it unwinds the duplex to provide templates for DNA polymerases. However, the molecular basis of the helicase-polymerase coupling is not fully understood. The recently elucidated T7 replisome structure suggests that the helicase and polymerase sandwich parental DNA and each enzyme pulls a daughter strand in opposite directions. Interestingly, the T7 polymerase, but not the helicase, carries the parental DNA with a positively charged cleft and stacks at the fork opening using a β-hairpin loop. Here, we created and characterized T7 polymerases each with a perturbed β-hairpin loop and positively charged cleft. Mutations on both structural elements significantly reduced the strand-displacement synthesis by T7 polymerase but had only a minor effect on DNA synthesis performed against a linear DNA substrate. Moreover, the aforementioned mutations eliminated synergistic helicase-polymerase binding and unwinding at the DNA fork and processive fork progressions. Thus, our data suggested that T7 polymerase plays a dominant role in helicase-polymerase coupling and replisome progression. Full article

(This article belongs to the Special Issue Mechanisms of DNA Replication Fork Progression, Stalling, and Rescue)

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13 pages, 1618 KiB

Open AccessArticle

Genetic Study of Four Candidate Holliday Junction Processing Proteins in the Thermophilic Crenarchaeon Sulfolobus acidocaldarius

by Ryo Matsuda, Shoji Suzuki and Norio Kurosawa

Int. J. Mol. Sci. 2022, 23(2), 707; https://doi.org/10.3390/ijms23020707 - 9 Jan 2022

Cited by 2 | Viewed by 1748

Abstract

Homologous recombination (HR) is thought to be important for the repair of stalled replication forks in hyperthermophilic archaea. Previous biochemical studies identified two branch migration helicases (Hjm and PINA) and two Holliday junction (HJ) resolvases (Hjc and Hje) as HJ-processing proteins; however, due [...] Read more.

Homologous recombination (HR) is thought to be important for the repair of stalled replication forks in hyperthermophilic archaea. Previous biochemical studies identified two branch migration helicases (Hjm and PINA) and two Holliday junction (HJ) resolvases (Hjc and Hje) as HJ-processing proteins; however, due to the lack of genetic evidence, it is still unclear whether these proteins are actually involved in HR in vivo and how their functional relation is associated with the process. To address the above questions, we constructed hjc-, hje-, hjm-, and pina single-knockout strains and double-knockout strains of the thermophilic crenarchaeon Sulfolobus acidocaldarius and characterized the mutant phenotypes. Notably, we succeeded in isolating the hjm- and/or pina-deleted strains, suggesting that the functions of Hjm and PINA are not essential for cellular growth in this archaeon, as they were previously thought to be essential. Growth retardation in Δpina was observed at low temperatures (cold sensitivity). When deletion of the HJ resolvase genes was combined, Δpina Δhjc and Δpina Δhje exhibited severe cold sensitivity. Δhjm exhibited severe sensitivity to interstrand crosslinkers, suggesting that Hjm is involved in repairing stalled replication forks, as previously demonstrated in euryarchaea. Our findings suggest that the function of PINA and HJ resolvases is functionally related at lower temperatures to support robust cellular growth, and Hjm is important for the repair of stalled replication forks in vivo. Full article

(This article belongs to the Special Issue Mechanisms of DNA Replication Fork Progression, Stalling, and Rescue)

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13 pages, 2873 KiB

Open AccessArticle

A Complexed Crystal Structure of a Single-Stranded DNA-Binding Protein with Quercetin and the Structural Basis of Flavonol Inhibition Specificity

by En-Shyh Lin, Ren-Hong Luo and Cheng-Yang Huang

Int. J. Mol. Sci. 2022, 23(2), 588; https://doi.org/10.3390/ijms23020588 - 6 Jan 2022

Cited by 16 | Viewed by 2293

Abstract

Single-stranded DNA (ssDNA)-binding protein (SSB) plays a crucial role in DNA replication, repair, and recombination as well as replication fork restarts. SSB is essential for cell survival and, thus, is an attractive target for potential antipathogen chemotherapy. Whether naturally occurring products can inhibit [...] Read more.

Single-stranded DNA (ssDNA)-binding protein (SSB) plays a crucial role in DNA replication, repair, and recombination as well as replication fork restarts. SSB is essential for cell survival and, thus, is an attractive target for potential antipathogen chemotherapy. Whether naturally occurring products can inhibit SSB remains unknown. In this study, the effect of the flavonols myricetin, quercetin, kaempferol, and galangin on the inhibition of Pseudomonas aeruginosa SSB (PaSSB) was investigated. Furthermore, SSB was identified as a novel quercetin-binding protein. Through an electrophoretic mobility shift analysis, myricetin could inhibit the ssDNA binding activity of PaSSB with an IC₅₀ of 2.8 ± 0.4 μM. The effect of quercetin, kaempferol, and galangin was insignificant. To elucidate the flavonol inhibition specificity, the crystal structure of PaSSB complexed with the non-inhibitor quercetin was solved using the molecular replacement method at a resolution of 2.3 Å (PDB entry 7VUM) and compared with a structure with the inhibitor myricetin (PDB entry 5YUN). Although myricetin and quercetin bound PaSSB at a similar site, their binding poses were different. Compared with myricetin, the aromatic ring of quercetin shifted by a distance of 4.9 Å and an angle of 31^o for hydrogen bonding to the side chain of Asn108 in PaSSB. In addition, myricetin occupied and interacted with the ssDNA binding sites Lys7 and Glu80 in PaSSB whereas quercetin did not. This result might explain why myricetin could, but quercetin could not, strongly inhibit PaSSB. This molecular evidence reveals the flavonol inhibition specificity and also extends the interactomes of the natural anticancer products myricetin and quercetin to include the OB-fold protein SSB. Full article

(This article belongs to the Special Issue Mechanisms of DNA Replication Fork Progression, Stalling, and Rescue)

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19 pages, 21372 KiB

Open AccessArticle

Delineation of the Ancestral Tus-Dependent Replication Fork Trap

by Casey J. Toft, Morgane J. J. Moreau, Jiri Perutka, Savitri Mandapati, Peter Enyeart, Alanna E. Sorenson, Andrew D. Ellington and Patrick M. Schaeffer

Int. J. Mol. Sci. 2021, 22(24), 13533; https://doi.org/10.3390/ijms222413533 - 16 Dec 2021

Cited by 4 | Viewed by 3058

Abstract

In Escherichia coli, DNA replication termination is orchestrated by two clusters of Ter sites forming a DNA replication fork trap when bound by Tus proteins. The formation of a ‘locked’ Tus–Ter complex is essential for halting incoming DNA replication forks. However, [...] Read more.

In Escherichia coli, DNA replication termination is orchestrated by two clusters of Ter sites forming a DNA replication fork trap when bound by Tus proteins. The formation of a ‘locked’ Tus–Ter complex is essential for halting incoming DNA replication forks. However, the absence of replication fork arrest at some Ter sites raised questions about their significance. In this study, we examined the genome-wide distribution of Tus and found that only the six innermost Ter sites (TerA–E and G) were significantly bound by Tus. We also found that a single ectopic insertion of TerB in its non-permissive orientation could not be achieved, advocating against a need for ‘back-up’ Ter sites. Finally, examination of the genomes of a variety of Enterobacterales revealed a new replication fork trap architecture mostly found outside the Enterobacteriaceae family. Taken together, our data enabled the delineation of a narrow ancestral Tus-dependent DNA replication fork trap consisting of only two Ter sites. Full article

(This article belongs to the Special Issue Mechanisms of DNA Replication Fork Progression, Stalling, and Rescue)

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23 pages, 4132 KiB

Open AccessArticle

Characterization of the Chimeric PriB-SSBc Protein

by En-Shyh Lin, Yen-Hua Huang and Cheng-Yang Huang

Int. J. Mol. Sci. 2021, 22(19), 10854; https://doi.org/10.3390/ijms221910854 - 7 Oct 2021

Cited by 11 | Viewed by 2473

Abstract

PriB is a primosomal protein required for the replication fork restart in bacteria. Although PriB shares structural similarity with SSB, they bind ssDNA differently. SSB consists of an N-terminal ssDNA-binding/oligomerization domain (SSBn) and a flexible C-terminal protein–protein interaction domain (SSBc). Apparently, the largest [...] Read more.

PriB is a primosomal protein required for the replication fork restart in bacteria. Although PriB shares structural similarity with SSB, they bind ssDNA differently. SSB consists of an N-terminal ssDNA-binding/oligomerization domain (SSBn) and a flexible C-terminal protein–protein interaction domain (SSBc). Apparently, the largest difference in structure between PriB and SSB is the lack of SSBc in PriB. In this study, we produced the chimeric PriB-SSBc protein in which Klebsiella pneumoniae PriB (KpPriB) was fused with SSBc of K. pneumoniae SSB (KpSSB) to characterize the possible SSBc effects on PriB function. The crystal structure of KpSSB was solved at a resolution of 2.3 Å (PDB entry 7F2N) and revealed a novel 114-GGRQ-117 motif in SSBc that pre-occupies and interacts with the ssDNA-binding sites (Asn14, Lys74, and Gln77) in SSBn. As compared with the ssDNA-binding properties of KpPriB, KpSSB, and PriB-SSBc, we observed that SSBc could significantly enhance the ssDNA-binding affinity of PriB, change the binding behavior, and further stimulate the PriA activity (an initiator protein in the pre-primosomal step of DNA replication), but not the oligomerization state, of PriB. Based on these experimental results, we discuss reasons why the properties of PriB can be retrofitted when fusing with SSBc. Full article

(This article belongs to the Special Issue Mechanisms of DNA Replication Fork Progression, Stalling, and Rescue)

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Review

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16 pages, 2735 KiB

Open AccessReview

The Biochemical Mechanism of Fork Regression in Prokaryotes and Eukaryotes—A Single Molecule Comparison

by Piero R. Bianco

Int. J. Mol. Sci. 2022, 23(15), 8613; https://doi.org/10.3390/ijms23158613 - 3 Aug 2022

Cited by 4 | Viewed by 2338

Abstract

The rescue of stalled DNA replication forks is essential for cell viability. Impeded but still intact forks can be rescued by atypical DNA helicases in a reaction known as fork regression. This reaction has been studied at the single-molecule level using the Escherichia [...] Read more.

The rescue of stalled DNA replication forks is essential for cell viability. Impeded but still intact forks can be rescued by atypical DNA helicases in a reaction known as fork regression. This reaction has been studied at the single-molecule level using the Escherichia coli DNA helicase RecG and, separately, using the eukaryotic SMARCAL1 enzyme. Both nanomachines possess the necessary activities to regress forks: they simultaneously couple DNA unwinding to duplex rewinding and the displacement of bound proteins. Furthermore, they can regress a fork into a Holliday junction structure, the central intermediate of many fork regression models. However, there are key differences between these two enzymes. RecG is monomeric and unidirectional, catalyzing an efficient and processive fork regression reaction and, in the process, generating a significant amount of force that is used to displace the tightly-bound E. coli SSB protein. In contrast, the inefficient SMARCAL1 is not unidirectional, displays limited processivity, and likely uses fork rewinding to facilitate RPA displacement. Like many other eukaryotic enzymes, SMARCAL1 may require additional factors and/or post-translational modifications to enhance its catalytic activity, whereas RecG can drive fork regression on its own. Full article

(This article belongs to the Special Issue Mechanisms of DNA Replication Fork Progression, Stalling, and Rescue)

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20 pages, 2862 KiB

Open AccessReview

Role and Regulation of Pif1 Family Helicases at the Replication Fork

by Emory G. Malone, Matthew D. Thompson and Alicia K. Byrd

Int. J. Mol. Sci. 2022, 23(7), 3736; https://doi.org/10.3390/ijms23073736 - 29 Mar 2022

Cited by 6 | Viewed by 3741

Abstract

Pif1 helicases are a multifunctional family of DNA helicases that are important for many aspects of genomic stability in the nucleus and mitochondria. Pif1 helicases are conserved from bacteria to humans. Pif1 helicases play multiple roles at the replication fork, including promoting replication [...] Read more.

Pif1 helicases are a multifunctional family of DNA helicases that are important for many aspects of genomic stability in the nucleus and mitochondria. Pif1 helicases are conserved from bacteria to humans. Pif1 helicases play multiple roles at the replication fork, including promoting replication through many barriers such as G-quadruplex DNA, the rDNA replication fork barrier, tRNA genes, and R-loops. Pif1 helicases also regulate telomerase and promote replication termination, Okazaki fragment maturation, and break-induced replication. This review highlights many of the roles and regulations of Pif1 at the replication fork that promote cellular health and viability. Full article

(This article belongs to the Special Issue Mechanisms of DNA Replication Fork Progression, Stalling, and Rescue)

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14 pages, 2071 KiB

Open AccessReview

Strengths and Weaknesses of Cell Synchronization Protocols Based on Inhibition of DNA Synthesis

by Anna Ligasová and Karel Koberna

Int. J. Mol. Sci. 2021, 22(19), 10759; https://doi.org/10.3390/ijms221910759 - 5 Oct 2021

Cited by 14 | Viewed by 9502

Abstract

Synchronous cell populations are commonly used for the analysis of various aspects of cellular metabolism at specific stages of the cell cycle. Cell synchronization at a chosen cell cycle stage is most frequently achieved by inhibition of specific metabolic pathway(s). In this respect, [...] Read more.

Synchronous cell populations are commonly used for the analysis of various aspects of cellular metabolism at specific stages of the cell cycle. Cell synchronization at a chosen cell cycle stage is most frequently achieved by inhibition of specific metabolic pathway(s). In this respect, various protocols have been developed to synchronize cells in particular cell cycle stages. In this review, we provide an overview of the protocols for cell synchronization of mammalian cells based on the inhibition of synthesis of DNA building blocks—deoxynucleotides and/or inhibition of DNA synthesis. The mechanism of action, examples of their use, and advantages and disadvantages are described with the aim of providing a guide for the selection of suitable protocol for different studied situations. Full article

(This article belongs to the Special Issue Mechanisms of DNA Replication Fork Progression, Stalling, and Rescue)

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